Liposomes have long been considered possible vehicles for delivery of therapeutic agents intracellularly. To date, however, success in achieving intracellular delivery of a liposome-entrapped agent has been limited for a variety of reasons. One reason is that liposomes, after systemic administration to the bloodstream, are rapidly removed from circulation by the reticuloendothelial system. Another reason is the inherent difficulty in delivering a molecule, in particular a large or a charged molecule, into the cellular cytoplasm and/or the nucleus.
One approach to improving intracellular delivery of liposome-entrapped agents is to extend the blood circulation lifetime of the liposome by including polyethyleneglycol (PEG) derivatized lipids in the liposome bilayer membrane (see, for example, U.S. Pat. No. 5,013,556). By extending the length of time that the liposomes remain in the bloodstream, the opportunity for uptake by a cell improves.
Another approach to improving intracellular delivery of liposome-entrapped agents is to provide a targeting moiety or ligand on the liposome (Klibanov et al., J. Liposome Res., 2(3):321 (1992)). Binding of the targeting moiety to a receptor on a target cell improves the chance of intracellular uptake of the liposome and its entrapped agent.
Also described in the art are liposomes capable of fusion with a target cell (U.S. Pat. No. 5,891,468). Fusogenic liposomes typically include a hydrophobic segment extending from the liposomes' outer surfaces for penetration into a target cell membrane.
Liposomes that destabilize under mildly acidic conditions, so-called ‘pH-sensitive’ liposomes, have also been described as an approach to intracellular delivery of an entrapped agent (Slepushkin et al., J. Biol. Chem., 272(4):2382 (1997); Wang et al., Proc. Natl. Acad. Sci., 84:7851 (1987), Liu et al., Biochim. Biophys. Acta, 981:254 (1989)). These liposomes are primarily composed of a lipid, such as dioleoylphosphatidylethanolamie (DOPE), that forms a lipid bilayer in a defined pH range. Outside this pH range, the lipid bilayer destabilizes. After such liposomes enter cells via endocytosis, the acidic pH inside the endosomes causes the pH-sensitive liposomes to destabilize and release the entrapped agent.
Because pH-sensitive liposomes, like “conventional”, “non-pH-sensitive liposomes”, have short circulation lifetimes, addition of PEG-derivatized lipids to extend the blood circulation time has been proposed (Slepushkin et al.). However, addition of PEG-derivatized lipids attenuates the pH-sensitivity of the liposomes, resulting in a loss of the desired rapid destabilization of the liposome bilayer and accompanying rapid release of the entrapped agent into the cell.
One approach to providing pH-sensitive liposomes having a long blood circulation lifetime and retaining the ability of the liposome to rapidly destabilize is the use PEG-derivatized lipids where the PEG is attached to the lipid by a chemically-cleavable linkage, such as a disulfide (Kirpotin et al., FEBS Letters, 388:115 (1996)). It is desirable, however, that the destabilization occurs in the cell to achieve a high intracellular concentration of the liposome-entrapped agent. The long-circulating, pH-sensitive liposomes described by Kirpotin et al. (Id.) were designed to destabilize extracellularly by release of the PEG chains. This approach suffers from the disadvantage of releasing the entrapped liposome contents extracellularly.
Accordingly, there remains a need in the art for a liposome composition capable of specific binding to a target cell accompanied by rapid intracellular release of its entrapped agent.